PIF2024 - Understanding DNA-end Processing and Joining using Single-Molecule Biophysical Methods - (PID2023-146255NB-I00)

A remarkable set of protein machines safeguards the integrity of our genome, continuously under thread from various factors. Central to DNA repair and maintenance are the homologous recombination (HR) and non-homologous end-joining (NHEJ) pathways, which are composed of a series of sequential processes aimed to repair potential DNA damage. In this project, we will focus on two critical processes within these pathways: the processing of DNA ends in HR and the role of lncRNAs in the NHEJ pathway. Our work will be centred around two set of proteins, CtIP and MRN, and Ku and lncRNAs. The human DNA repair factor CtIP helps to initiate the resection of double-stranded DNA breaks for repair by HR. It works together with the MRN complex. However, the molecular mechanisms governing CtIP’s function remains unknown, mainly due to its intrinsically disordered structure. Ku is a heterodimer formed by Ku70 and Ku80 subunits, and recent studies have shown that it interacts with specific long non-coding RNAs, enhancing the efficiency of DNA end joining. We will investigate how these proteins participate in the process of DNA repair using the arsenal of single molecule methods developed and available in our group. Furthermore, our research extends to the development of instruments and methods, particularly to a combined AFM-TIRF microscope that we aim to push to state-of-the-art technology.